Interleukin secretion by B cell lines and splenic B cells stimulated with calcium ionophore and phorbol ester

A B cell lymphoma A20.2J and splenic B cells produced an active material to support the proliferation of an interleukin 2 (IL-2)-dependent T cell line, CTLL-2, by stimulation with both calcium ionophore A23187 and phorbol myristate acetate (PMA). Although the production of the active material was induced by stimulation with A23187 alone in A20.2J cells, both A23187 and PMA were essential for the stimulation of splenic B cells. Neither A20.2J cells nor splenic B cells produced the active material by stimulation with PMA alone. The production was inversely proportional to the concentration of fetal calf serum in culture medium. The active material produced by B cells was indicated to be IL-2 and not B cell-stimulating factor 1 (BSF-1) for the following reasons: 1) the proliferation of CTLL-2 cells in the presence of active material was inhibited by the inclusion of anti-IL-2 receptor or anti-IL-2 in culture medium but not by anti-BSF-1; 2) the material showed no co-mitogenic activity to purified splenic B cells with anti-immunoglobulins and did not support the proliferation of FDC-P2 which are known to grow in the presence of BSF-1; and 3) IL-2 mRNA could be detected in A20.2J and splenic B cells stimulated with A23187 and PMA in Northern blot analysis. Some B cell hybridomas were also shown to produce IL-2 by similar stimulation to A20.2J. Splenic B cells as well as A20.2J cells were able to produce IL-2 by stimulation with anti-immunoglobulins. These results suggest that under certain conditions IL-2 can be produced by splenic B cells, at least some subsets of B cells, and B cell lines.     

Blocking with phorbol ester, a protein kinase C activator, of receptor-dependent platelet calcium channels

The regulation of receptor-operated calcium channels of human platelets by phospholipid-dependent, Ca2+- and diacylglycerol-activated protein kinase C was studied. In order to induce the activation of endogenous protein kinase C, a cell-penetrable structural diacylglycerol analog, 4 beta-phorbol 12 beta-myristate-13 alpha-acetate (FMA), was used. Using two independent approaches, i. e., the fluorescent probe for Ca2+, quin-2, and 45Ca2+ absorption technique, it was demonstrated that FMA (10(-10) - 10(-8) g/ml) blocks Ca2+ influx into the platelets induced by aggregation factors, e. g., ADP, vasopressin, platelet activating factor, thrombin and thromboxane A2 receptor agonist U46619. The half-maximum inhibition of the receptor-sensitive influx of Ca2+ was observed at (3-6) X 10(-10) g/ml of FMA. Under physiological conditions, protein kinase C is activated with an increase in Ca2+ concentration in the cytoplasm in the presence of diacylglycerol. Since the above-mentioned inducers besides Ca2+ influx stimulate diacylglycerol synthesis, it was assumed that the activation of protein kinase C triggers a negative feedback mechanism which blocks the receptor-operated calcium channels.     

Inhibition of thymine dimer excision by the phorbol ester, phorbol myristate acetate

The effect of the promoting agent, phorbol myristate acetate, on repair of UV-induced damage in HeLa cells was studied. The agent decreased survival and subsequent colony-forming ability of irradiated cells and inhibited removal of UV-induced thymine-containing dimers from DNA of irradiated cells.     

Synergistic action of A23187 and phorbol ester on human B cell activation

We have investigated the existence of a synergy occurring between the calcium ionophore A23187 and phorbol myristic acetate (PMA) with respect to human B cell proliferation and differentiation. The combination of A23187 (250 to 500 nM) with nonmitogenic concentrations of PMA (1 to 3 ng/ml) resulted in a strong proliferative response in human tonsillar, spleen, and peripheral blood B cells. This proliferation could not be blocked by anti-Tac antibody at concentrations that effectively inhibited T cell proliferation under similar culture conditions, suggesting that IL 2 and its receptor are not involved in B cell proliferation in this system. During a 3-day culture period, A23187 (500 nM) did not activate B cells in terms of changes in cell size or in the expression of transferrin receptor, HLA-DR, and Tac antigen. PMA at a nonmitogenic concentration (3 ng/ml) enhanced the expression of the first two markers. Combination of the ionophore with PMA induced the occurrence of Tac and further increased the expression of transferrin receptor and HLA-DR. A23187 similarly enhanced the PMA-mediated increase in cell size. PMA and A23187 did not induce differentiation to lg production. However, when cells were prestimulated with a combination of the two agents and were recultured in the presence of a preparation containing B cell differentiation factor, a strong increase in IgM, IgG, and IgA production was found. We conclude that PMA and A23187 synergistically trigger intracellular events in human B cells, leading to proliferation and to responsiveness to differentiation factors.     

Endothelin/sarafotoxin receptor induced phosphoinositide turnover: effects of pertussis and cholera toxins and of phorbol ester

The induction of phosphoinositide hydrolysis (PI) by endothelin/sarafotoxin (ET/SRTX) receptors in rat heart myocytes was investigated by the use of bacterial toxins as well as a phorbol ester. Both pertussis- and choleratoxin enhanced the stimulation of PI hydrolysis. Phorbol ester treatment of the myocytes for short periods distinguished between two types of PI-hydrolysis, the one induced by endothelins and the other by sarafotoxins. The possible mediation of G-protein (s) in the induction by ET/SRTX receptors of PI-hydrolysis is discussed.     

The effect of a phorbol ester on amphibian myogenesis

The effect of a phorbol ester (TPA) on myogenesis of urodelian embryo has been investigated. Presumptive somites were extirpated from mid-neurula, the stage when they have already attained certain degree of determination. The explants were treated with TPA (10 ng/ml in Steinberg solution with the addition of 10% Leibovitz's L-15) for 4 days, the period during which the frequency and size of gap junctions in normal myogenic cells are at the maximum. After the treatment many cells became dispersed and isolated while others spread out and remained connected in the form of a sheet. During further culture for 6 days in the solution without TPA myogenesis was blocked. Both the dispersed cells and the cells in a sheet remained at the earliest stage of muscle differentiation with irregular shape or in the form of myoblast, while cells of the control series formed myotubes containing bundles of myofibrils. The result suggests that the blockage of myogenesis is the consequence of interruption of junctional communication evoked by TPA treatment.     

Regulation of cell division of mature B cells by ionomycin and phorbol ester.

The growth of a human B lymphoma cell line B104, an experimental model for mature B cells, was inhibited by ionomycin but not 12-O-tetradecanoylphorbol-13-acetate (TPA). Ionomycin inhibited B104 cells from entering into the M phase of the cell cycle without affecting DNA synthesis. The inhibition of cell division of B104 cells by ionomycin occurred within 24 h after stimulation. Because such a mode of action resembles that of anti-IgM antibodies, signals transduced by Ca2+ may be responsible for the inhibition of cell division of B104 cells by anti-IgM antibodies. Indeed, EGTA suppressed the inhibition of cell division of B104 cells caused not only by ionomycin, but also by anti-IgM antibody. Although TPA itself did not have any ability to promote the growth of B104 cells, it could cancel the inhibition of cell division of B104 cells by ionomycin and increase the proportion of B104 cells entering into the M phase of the cell cycle. Staphylococcus aureus Cowan I causes the greatest proliferation of normal human peripheral blood B cells during the period from 48 to 72 h after stimulation. When ionomycin was added to S. aureus Cowan I-stimulated peripheral blood B cells at 48 h of culture, it inhibited cell division during this period without affecting DNA synthesis. In the presence of TPA, this activity of ionomycin was suppressed, and the proportion of M-phase cells increased. These results suggest that cell division of mature B cells is regulated by the signals mediated by Ca2+ and protein kinase C in a mode quite different from that of regulation of DNA synthesis.     

Human glycophorin A and B are encoded by separate, single copy genes coordinately regulated by a tumor-promoting phorbol ester

Human glycophorin A belongs to a family of structurally related cell surface glycoproteins which are expressed in erythroid cells. We have recently isolated several human glycophorin A-specific cDNA clones and have determined their nucleotide sequence (Siebert, P. D., and Fukuda, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1665-1669). In this report by using cDNA and exact sequence synthetic oligodeoxyribonucleotides as hybridization probes, we provide evidence that human glycophorin A and B are encoded by separate and distinct genes present as single copies in the human genome. Furthermore, we show that the expression of the glycophorin A and B genes are coordinately regulated by tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate.     

A phorbol ester and a daphnane ester stimulate a calcium-independent kinase activity from human mononuclear cells

TPA and a non-promoting, pro-inflammatory ester RX were used to stimulate the forms of PKC isolated from human mononuclear cells. Three peaks of kinase activity corresponding to gamma, beta and alpha PKC were stimulated by TPA in the presence and absence of calcium and/or phosphatidylserine (PS) but were not activated by RX. A fourth peak eluted at high phosphate concentration was activated by TPA and RX in the presence of PS and the absence of calcium. Activity in this fraction was labile to freezing and thawing and was inhibited by staurosporine.     

Newly synthesized dopamine ester derivatives and assessment of their antioxidant,antimicrobial and hemolytic activities

Preparation of dopamine derivatives was carried out as a response to the increasing demand for new lipophilized antioxidants in food, cosmetic and pharmaceutical industries. A large series of dopamine esters (DA-C₃ to DA-C_18:1) with increasing lipophilicity was synthesized using lipase from Candida antarctica (Novozyme 435) as a biocatalyst. The highest conversion yield (52.75%) was reached when caprylic acid (DA-C₈) was used as acyl donor. Synthesized compounds were purified and evaluated for their antioxidant activity using the DPPH and the ABTS tests. Results showed that esterification had little effect on radical-scavenging capacity. However, long chain fatty acid esters displayed higher protective effect of oil against oxidation at 70 °C as compared to the parent dopamine or to the BHT. The hemolytic activity of dopamine esters was studied. Middle chain length derivatives (DA-C₈ and DA-C₁₂) of dopamine and oleic acid derivative (DA-C_18:1) showed the highest hemolytic activity against human erythrocyte. The antimicrobial activities of dopamine esters were also evaluated using well diffusion and minimal inhibition concentration methods. Among all the tested compounds, DA-C₈ and DA-C₁₂ exhibited the highest antibacterial activities. These results open up potential applications by using dopamine derivatives as antioxidants and antimicrobial compounds in cosmetic, food and pharmaceutical industries.     

Calcium-dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter

The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two-phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase. Ionophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.     

Intracellular calcium redistribution and its relationship to fMet-Leu-Phe, leukotriene B4, and phorbol ester induced rabbit neutrophil degranulation

The addition of low concentrations (less than 10(-7) M) of the calcium ionophore A23187 to rabbit neutrophils releases the intracellular pool of calcium previously shown in radioactive steady-state and chlortetracycline fluorescence studies to be mobilized by chemotactic factors. A23187 at these concentrations elicits no functional responses from these cells. However, A23187, added before chemotactic factors such as fMet-Leu-Phe and leukotriene B4, inhibits the ability of the latter stimuli to induce, in the presence of cytochalasin B, an exocytotic release of the neutrophil's cytoplasmic granules. These results imply that the chemotactic-factor-induced release of intracellular calcium is a necessary event for the optimal activation of the neutrophils. Phorbol ester-induced neutrophil degranulation on the other hand is unaffected by exposure to A23187, thereby completely dissociating its mechanism of action from rises in cytoplasmic free calcium.     

Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3'-phosphorylation of phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.     

Anti-immunoglobulin and phorbol ester induce phosphorylation of proteins associated with the plasma membrane and cytoskeleton in murine B lymphocytes

Protein phosphorylations are rapidly induced in intact B cells by antibodies to surface immunoglobulin (anti-IgM) and by phorbol 12-myristate 13-acetate (PMA). A comparison of the molecular weight, isoelectric points, phosphopeptides, and phosphoamino acids of the phosphoproteins induced by anti-IgM and by PMA suggests that anti-IgM acts through the activation of protein kinase C. This conclusion is strengthened by the observation that prolonged treatment with PMA ablates the ability of anti-IgM to induce phosphorylation, presumably by depleting cellular protein kinase C. Furthermore, the effects of dibutyryl cyclic AMP on protein phosphorylation are quite distinct from the effects of anti-IgM. The six most prominent phosphoproteins induced by PMA, with approximate Mr values of 47, 55, 62, 68, 68, and 65-70 X 10(3), are associated with the plasma membrane. Of these, four are apparently associated with the cytoskeleton, suggesting that the phosphorylation of cytoskeletal proteins may be important events early in B cell activation. Examination of protein phosphorylation in cell lines derived from different tissues has identified one major B cell phosphoprotein (Mr 65-70 X 10(3), which is absent in T cells, and two phosphoproteins (Mr 55 and 68 X 10(3), which are observed in cells of hematopoietic origin but which are absent or uncommon in other cell types.     

Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody

The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture.     

Effect of a phorbol ester on basic surface properties of trichomonads

The effect of nanomolar concentrations of 12-O-tetradecanoil-phorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoaTrichomonas vaginalis andTritrichomonas foetus was evaluated by means of measurements of the parasites’ surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites’ net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.     

Actions of a tumor-promoting phorbol ester in a cloned rat pituitary cell

Tumor promoters, such as phorbol myristate acetate (PMA), facilitate carcinogenesis by mechanisms that may involve changes in intracellular Ca²⁺ metabolism and distribution of Ca²⁺, as well as activation of a Ca²⁺-and phospholipid-dependent protein kinase, referred to as protein kinase C. We compared the actions of PMA on GH₃ cloned pituitary cells with those of thyrotropin releasing hormone (TRH), an established Ca²⁺-mobilizing agent. The TRH treatment produced a⁴⁵Ca efflux, inhibited⁴⁵Ca uptake, diminished chlortetracycline fluorescence, and stimulated cAMP accumulation and protein synthesis in a Ca²⁺-dependent manner. Like TRH, PMA produced an efflux of⁴⁵Ca and inhibited⁴⁵Ca uptake; however, the phorbol ester stimulated cAMP accumulation and protein synthesis in the absence of external calcium and failed to alter chlortetracycline fluorescence. The TMB-8, a putative inhibitor of the mobilization of membrane-associated Ca²⁺, did not alter PMA-induced stimulation of protein synthesis. The results suggest that PMA-induced changes in Ca²⁺ metabolism are not caused by the mobilization of membrane-associated calcium. Alternative proposals are that PMA (1) inhibits Ca²⁺ influx and/or (2) mobilizes calcium from nonmembranous storage sites. Further study is needed to characterize the mechanism through which tumor-promoting phorbol esters influence Ca²⁺ metabolism and to ascertain the significance of changes in Ca²⁺ metabolism to cellular processes affected by these substances.     

Changes in fibroblast contractility, morphology, and adhesion in response to a phorbol ester tumor promoter

We have studied the effects of the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the contractility, locomotion, morphology, and adhesion of two mammalian fibroblastic cell lines. Using the silicone rubber substratum technique, we have found that the first observable response to the tumor promoter is a rapid weakening of cell contractility (8-15 min). This is followed by gradual morphological changes, characterized by a hyperextension of the cells' leading lamellae, which stretch out to an unlimited degree, and occasionally even detach from the cell bodies. Treated cells also become able to crawl onto hydrophobic substrata which are insufficiently adhesive to support the spreading of untreated fibroblasts. We suggest that both the hyperextension and the ability to spread on nonadhesive surfaces can be explained as consequences of the reduced contractility, and that this reduced contractility may also help to explain the increased invasiveness and loss of anchorage dependence by transformed cells.     

Purification and characterization of a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase from porcine spleen

A calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase was purified to apparent homogeneity from a Triton X-100 extract of an EGTA/EDTA-preextracted particulate fraction of porcine spleen by chromatography on S-Sepharose Fast Flow, phenyl-Sepharose Fast Flow, protamine-agarose, and Superdex 200. The enzyme had a Mr of 76,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (p76-kinase). A similar value (78,000) was obtained by gel filtration. The purified p76-kinase proved to be much more stable than the enzyme in crude preparations. Storage in a buffer containing 50 mM mercaptoethanol and 20% glycerol at -20 degrees C for at least 4 months caused less than 20% loss in enzyme activity. The enzyme exhibited a pH optimum of 8.3. The affinity of the novel enzyme for substrates and cofactors differed to some extent from that of conventional alpha, beta, gamma protein kinase C (PKC). p76-kinase did not respond to calcium, had a lower requirement for magnesium, and a higher affinity for histone III-S than PKC. Both the p76-kinase-catalyzed phosphorylation of histone III-S and the autophosphorylation of the enzyme could be activated by the phorbol ester TPA (or diacylglycerol) plus phosphatidyl serine, but not by calcium plus phosphatidyl serine. The stoichiometry of autophosphorylation suggested that fully phosphorylated p76-kinase contained two phosphoserine residues and one phosphothreonine residue. Like PKC, p76-kinase bound TPA with high affinity (KD = 9.6 nM). In the absence of TPA, various unsaturated fatty acids, particularly arachidonic acid, were more potent as activators of the enzyme than phosphatidyl serine. The p76-kinase was recognized by an antiserum raised against a delta PKC-specific peptide, but not by an alpha, beta, gamma PKC-specific antiserum. The previously described p82-kinase of mouse epidermis and spleen exhibiting the same properties as the p76-kinase did also react with the p76-kinase-specific antiserum.